Immunotherapy, including T-cell checkpoint inhibition, has rapidly changed the outcome of advanced cancers in a subset of patients. To extend benefit of these promising therapies to more patients, we must delineate predictive biomarkers and develop approaches to enhance the efficacy of checkpoint blockade. Our long-term goal is to define the contribution of tumor genetics to clinical benefit from checkpoint blockade immunotherapies and to use this information to develop better therapeutic modalities. Our group has shown that an elevated mutational burden correlates with benefit from therapy in melanoma patients treated with anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) therapies. For the proposed project, we have collected melanoma tumors (N=42) from pembrolizumab-treated patients and will perform the correlative genetics analyses for a Phase II trial of nivolumab ipilimumab in advanced ovarian cancer (planned accrual, N=96). Using these samples, in Aim 1 we will examine our working hypothesis that an elevated mutational burden increases the likelihood of response to checkpoint blockade in melanoma and ovarian cancers. We will then integrate T cell receptor (TCR) sequencing data available from the melanoma tumors and peripheral blood to investigate the hypothesis that TCR oligoclonal dominance will add to mutational burden as a predictor of response. In Aim 2, we will identify and clone neoantigen-specific T cells from melanoma patients treated with pembrolizumab, and then use these clones to investigate our hypothesis that neoantigen-specific T cells can be activated by homologous peptides. Finally, in Aim 3, we will characterize whether therapy-induced DNA damage can sensitize melanoma and ovarian cancer cell lines to checkpoint blockade therapy using in vitro and in vivo models of melanoma and ovarian cancer. These experiments will determine whether therapy-induced mutagenesis can sensitize tumors to checkpoint blockade therapy. The preclinical and correlative studies outlined in this proposal have the potential to (a) elucidate mechanisms of response to immunotherapy in two fatal cancers, and (b) provide data to begin to answer the clinically important question of whether non-immunogenic tumors can be sensitized to checkpoint blockade therapy by inducing mutations. The studies will be led by Dr. Alexandra Snyder, a junior faculty member at Memorial Sloan Kettering Cancer Center under the mentorship of Dr. Timothy Chan, a leading tumor geneticist; Dr. Jedd Wolchok, a renowned tumor immunologist; and Dr. Carol Aghajanian, a world leader in Gynecologic Oncology. To achieve her long-term goal of become an independent investigator, Dr. Snyder has assembled an advisory committee of leading scientists, and has developed a structured curriculum aimed at deepening her knowledge base so she can advance the field.